Total protein was extracted from HCC cells using RIPA lysis buffer (Invitrogen, Carlsbad, Calif, USA). Briefly, equal amounts of A quantity of approximately 30 μg of whole cell lysates per lane were separated using 10% SDS-PAGE and transferred on to immobilon PVDF membranes (Millipore, Corporation, Billerica, MA, USA). PVDF membranes were incubated with the primary antibodies of CDK6 (Abcam, 1:2000 dilution) and anti-GAPDH (Santa Cruz, 1:1,000 dilution). Blots were incubated with goat anti-rabbit secondary antibody conjugated to HRP (Abcam, 1:1000 dilution). PVDF membranes were visualized by a standard enhanced chemiluminescence procedure (Millipore, Billerica, MA, USA). The signals were analyzed using Image Lab software (Bio-Rad Laboratories, Inc., Hercules, CA). The band densities of each sample were normalized to the GAPDH band.
Standard enhanced chemiluminescence procedure
Manufactured by Merck Group
Sourced in United States
The Standard Enhanced Chemiluminescence Procedure is a laboratory equipment product that utilizes chemiluminescence, a process in which light is emitted from a chemical reaction. This equipment is designed to facilitate the detection and analysis of specific molecules or proteins in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Immobilon pvdf membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Radioimmunoprecipitation lysis buffer, by Thermo Fisher Scientific (1 mentions)
E cadherin, by Abcam (1 mentions)
N cadherin, by Abcam (1 mentions)
Cyclin d1, by Abcam (1 mentions)
C myc, by Abcam (1 mentions)
β catenin, by Abcam (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Merck Group (1 mentions)
Image pro plus, by Media Cybernetics (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
3 protocols using standard enhanced chemiluminescence procedure
1
Western Blot Analysis of CDK6 in HCC Cells
2
Western Blot Analysis of HCC Cell Proteins
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After protein extraction from HCC cells in radioimmunoprecipitation lysis buffer (Invitrogen), 10% SDS/PAGE was used to separate equal amounts of proteins, followed by the shifting of proteins to polyvinylidene difluoride membranes (Millipore Corporation, Billerica). The membranes were then detected with primary antibodies of SOX9, β‐catenin, Cyclin D1, c‐Myc, E‐cadherin, N‐cadherin (Abcam, UK) and GAPDH (Santa Cruz Biotechnology Inc., Dallas, TX, USA). Thereafter, blots were incubated with the horseradish peroxidase‐conjugated secondary antibody (Abcam). Visualization of blots was conducted by the standard enhanced chemiluminescence procedure (Millipore, Billerica). Analysis of signals was performed on image lab software (Bio‐Rad Laboratories, Inc.).
3
Protein Expression Analysis via Western Blot
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Total cell protein was extracted in Radio Immunoprecipitation Assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing a protease inhibitor cocktail (Roche, Mannheim, Germany). The protein extracts were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. After blocking, the membrane was probed with primary antibody against a-SMA (1:1000), FN (1:1000; Protein-Tech, Chicago, IL, USA), and GAPDH (1:2000; Millipore, Billerica, MA, USA), followed by the appropriate horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibody (Millipore). Specific bands were visualized by a standard enhanced chemiluminescence procedure (Millipore). The signals were analyzed using Image-Pro Plus (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA). The band density of each sample was normalized to the GAPDH band.
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